Yoshimichi Matsunaga

Relationship between NMO-antibody and anti-MOG antibody in optic neuritis.

Background: Damage to astrocytes by anti-aquaporin-4 antibody (AQP4-Ab), also known as NMO antibody, has been implicated as the cause of neuromyelitis optica. Myelin oligodendrocyte glycoprotein (MOG) is well known as the causative protein of multiple sclerosis (MS). MOG antigen is currently considered as a cause of optic neuritis (ON) associated with MS because immunization with MOG antigen derived from oligodendrocytes induces murine ON with myelitis. We investigated the relationship between NMO antibody (NMO-Ab) and anti-MOG antibody (MOG-Ab) and potential in patients with ON for recovery of vision. Methods: Thirty-three eyes of 23 patients with ON were studied. At presentation, serum NMO-Ab was measured by immunofluorescence using HEK 293 cells transfected with AQP4–GFP, and anti-MOG1–125 antibody was measured by enzyme-linked immunosorbent assay. MOG-Ab seropositivity was defined by comparing with MOG-Ab level obtained from 8 healthy normal subjects. Results: Eleven (47%) of 23 ON patients were NMO-Ab seropositive, while 8 (34%) of the 23 patients were MOG-Ab seropositive. Six (26%) of 23 patients were seropositive for both NMO-Ab and MOG-Ab. Ten (43%) of 23 patients were seronegative for both antibodies. Three (50%) of 6 eyes of patients seropositive for both antibodies did not respond to corticosteroid pulse therapy and plasmapheresis, and visual acuity remained unchanged. In the NMO-Ab(−)/MOG-Ab(−) group, visual acuity improved significantly (P < 0.0001). In the other 3 groups (NMO-Ab(+)/MOG-Ab(+), NMO-Ab(+)/MOG-Ab(−), and NMO-Ab(−)/MOG-Ab(+)), visual acuity did not change significantly (P = 0.53, 0.42, and 0.45, respectively). Conclusion: NMO-Ab and MOG-Ab could be potential biomarkers to determine visual prognosis in patients with ON.

Visual Functional and Histopathological Correlation in Experimental Autoimmune Optic Neuritis

PURPOSE To elucidate the correlation between visual threshold of optokinetic tracking (OKT), visual evoked potential (VEP), and histopathology at different time points after induction of experimental autoimmune optic neuritis (EAON). METHODS EAON was induced in C57BL/6 mice by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide. OKT and VEP were measured on days 7, 14, 21, 28, and 42 postimmunization. After VEP measurements, the mice were killed and their eyes were enucleated for histopathological studies. Immunohistochemical staining was performed using cell-specific markers for characterization of cells in the optic nerve: CD3 (T cells), Iba-1 (microglia), MBP (myelin basic protein), and neurofilament (axons). RESULTS Functionally, OKT threshold decreased as early as day 7, and VEP latency was significantly prolonged on day 21. Axon degeneration was observed as early as day 14. Activated microglia infiltration was also observed on day 14, before T cell infiltration, which peaked on day 21. Demyelination, confirmed by MBP staining, was observed on day 21. CONCLUSIONS Microglial infiltration in the optic nerve coincided with decline in OKT threshold and preceded VEP latency prolongation, while VEP latency prolongation coincided with T cell infiltration and demyelination of the optic nerve. These findings may contribute to understanding of the pathophysiology of optic neuritis and future development of more effective therapeutic strategy for refractory optic neuritis.

Interleukin-10 gene-transfected mature dendritic cells suppress murine experimental autoimmune optic neuritis.

PURPOSE We have reported that calcitonin gene-related peptide gene-transfected mature dendritic cells (mDC) suppress murine experimental autoimmune optic neuritis (EAON) and experimental autoimmune encephalitis (EAE) via interleukin-10 (IL-10) production. In our study, we examined whether IL-10-transfected mDC prevent development of EAON and EAE. METHODS A plasmid expressing mouse IL-10 was constructed and used to transfect C57BL/6 mouse bone marrow-derived mDC by electroporation methods. C57BL/6 mice (with or without GFP expression) were immunized with myelo-oligodendrocyte glycoprotein₃₅₋₅₅ (MOG₃₅₋₅₅), and injected intravenously with IL-10-transfected mDC either in the induction or effector phase. RESULTS When IL-10-transfected mDC were injected in the induction phase, EAE developed clinically in 60% of mice in the IL-10-transfected group compared to 100% in the mock-transfected group (P < 0.05), and mean pathologic score for EAON was 1.1 in the IL-10-transfected group compared to 2.1 in the mock-transfected group (P < 0.05). When IL-10-transfected mDC were injected in the effector phase, mean EAE clinical scores were not significantly different between the two groups (2.0 vs. 3.0), while the mean EAON pathologic score was lower in the IL-10-transfected group compared to the mock-transfected group (1.0 vs. 2.7, P < 0.05). Delayed hypersensitivity was suppressed significantly in the IL-10-transfected group. Interestingly, the proportions of CD80/86⁺ and MHC class II⁺ cells decreased significantly (P < 0.05), whereas Foxp3⁺ cells increased significantly in the spleen and lymph node in the IL-10-transfected group by flow cytometry analysis. Immunohistochemical analysis demonstrated the localization of IL-10-transfected GFP-expressing mDC not only in the spleen and lymph nodes but also in the inflamed optic nerve. CONCLUSIONS Treatment with IL-10-expressing mDC was effective in suppressing the development of EAON and EAE.

Suppression of murine experimental autoimmune optic neuritis by mature dendritic cells transfected with calcitonin gene-related Peptide gene.

PURPOSE Calcitonin gene-related peptide (CGRP) exhibits prominent anti-inflammatory actions. We examined whether CGRP-transfected dendritic cells (DC) prevent the development of experimental autoimmune optic neuritis (EAON) and experimental autoimmune encephalomyelitis (EAE). METHODS A human CGRP-expressing plasmid was constructed, and used to transfect C57BL/6 mouse bone marrow-derived matured DC (mDC) by electroporation METHODS Transfection efficiency was 50% with 80% cell viability. C57BL/6 mice were immunized with myelo-oligodendrocyte glycoprotein 35-55, and injected intravenously with CGRP-expressing mDC (CGRP gene-transfected group) or mock-transfected mDC (mock-transfected group) at the induction or effector phase. EAE was diagnosed clinically and EAON was assessed histopathologically. Delayed hypersensitivity was measured. Supernatants of spleen cell cultures were assayed for cytokines using ELISA. The CD4(+)CD25(+)Foxp3(+) fraction in spleen cells was analyzed using flow cytometry. RESULTS For gene therapy in the induction phase, EAE developed in 50% of mice in the CGRP-transfected group compared with 80% in the mock-transfected group, and the mean pathological score for EAON was 1 in the CGRP-transfected group compared with 2 in the mock-transfected group (P < 0.05). For gene therapy in the effector phase, the mean EAE clinical score (1.5 vs. 3.0) and mean EAON pathological score (1.0 vs. 2.0) were both lower in the CGRP-transfected group compared with the mock-transfected group (P < 0.05). Delayed hypersensitivity was suppressed significantly in the CGRP-transfected group. IL-10 production by spleen cells in the CGRP-transfected group increased independent of MOG concentration, compared with the mock-transfected group. Interestingly, the proportion of CD4(+)CD25(+)Foxp3(+) cells increased significantly (P < 0.05) in the CGRP-transfected group compared with the mock-transfected group. CONCLUSIONS Gene therapy with CGRP-expressing mDC was effective in suppressing the development of EAON and EAE.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Suppression of experimental autoimmune optic neuritis by the novel agent fingolimod

Purpose: Fingolimod is an immunomodulating agent that has been approved for the treatment of multiple sclerosis. Fingolimod-phosphate is an antagonist of sphingosine-1-phosphate receptor and known to act by preventing infiltration of autoreactive lymphocytes into the central nervous system. In this study, we investigated whether fingolimod prevents experimental autoimmune optic neuritis (EAON). Methods: EAON was induced by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein–derived peptide 35–55 (MOG-p). After MOG-p immunization, fingolimod was administered intragastrically from day 1 (entire phase study) or from day 9 (effector phase study) until day 35. Visual acuity of the mice was measured using OptoMotry on days 7, 14, 21, 28, and 35 after immunization. On day 35 after immunization, the mice were killed and eyes and entire length of the optic nerves were submitted for histopathologic evaluation. Results: In the positive control group, visual acuity decreased markedly from approximately day 14 after immunization, reaching a nadir on day 21. In the fingolimod-treated groups in both entire phase and effector phase studies, there was only minimal decline in visual acuity on day 14 after immunization, and mild deterioration on day 21, followed by recovery. Histopathologic study showed that fingolimod given throughout the entire phase or only from the effector phase suppressed murine EAON. Immunohistochemical study for neurofilament demonstrated no irregularity of the linear structure of the optic nerve in the fingolimod-treated mice compared with the positive control group. Conclusion: Fingolimod ameliorated EAON even when started after optic neuritis had developed. Further study is warranted to examine whether these findings are applicable to human disease.

Refractory Eosinophilic Granulation Tissue of the Palpebral Conjunctiva

A 32-year-old Japanese man complained of a mass in his right upper palpebral conjunctiva and presented with symptoms of epiphora, foreign body sensation in the eye, and pruritus. Prior to formation of the mass, he had suffered a chemical ocular injury with raw concrete in November 2003. He also had atopic dermatitis, for which he was being treated with topical medication. His visual acuity and intraocular pressure were normal. Slit-lamp examination revealed a coralloid peduncular mass measuring 10 mm × 10 mm in his right upper palpebral conjunctiva (Fig. 1A). The results of a blood test were normal, except for an increase in nonspecifi c IgE to 2100 IU mg/dl. A conjunctival bacterial culture yielded negative results. Antiallergic, antibiotic, and corticosteroid eye drops proved ineffective; therefore, resection with cautery was performed in August 2004. Two months later, the mass recurred in the right palpebral conjunctiva, and the patient became symptomatic. Another surgery was performed with cryopexy and subconjunctival injection with betamethasone. After 2 months, a third resection was performed, following which the mass recurred yet again. One month later, surgery was performed for the fourth time, and tarsal subconjunctival injections of triamcinolone were administered after the resection. After the fourth resection, eye drops containing 0.04% mitomycin C were used for a week, after which they were discontinued because the patient developed superfi cial punctuate keratopathy. After the fi fth resection, 30 mg prednisone was orally administered for a week, but the medication was discontinued because of poor compliance. After the seventh resection, gentamicin, betamethasone, and triamcinolone were injected into the tarsal subconjunctival space. Eye drops containing 1% cyclosporine were administered as well, but had to be discontinued because the patient developed superfi cial punctuate keratopathy. Despite the alterations in the treatment, granulation tissue recurred within as little as 2 weeks (Fig. 1C), LETTERS